Detection method of aminoglycoside drug intermediate
The quality of 1,3,2',6',3''-Van-N-p-toluenesulfonylkanamycin B intermediate was detected by HPLC chromatography, which solved the problem of detection difficulty in the prior art and achieved the realization of The fast and accurate detection effect ensures the quality and yield of the final product Dibeccastar.
Patent Information
- Application Number
- CN202311036520.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-08-17
- Publication Date
- 2025-02-21
AI Technical Summary
The prior art is difficult to quickly and accurately detect the mass of 1,3,2',6',3'-Van-N-p-toluenesulfonylkanamycin B intermediates, affecting the quality of the final product Dibecacin and Yield.
Using HPLC chromatography, the specific column, mobile phase composition and detection wavelength can achieve rapid and accurate 1,3,2',6',3'-Van-N-p-toluenesulfonylkanamycin B Test.
This method can quickly and accurately separate and quantitatively analyze 1,3,2',6',3'-Vent-N-p-toluenesulfonylkanamycin B, with detection limit of 0.3 µg/mL, and the principal component repeats The RSD of 0.23% ensures the purity of the intermediate and the quality of the final product.
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Abstract
Description
Technical Field
[0001] The invention relates to a detection method, in particular to a detection method for an aminoglycoside drug intermediate. Background Art
[0002] 3′,4′-dideoxy-kanamycin B, also known as dibekacin, is a marketed semi-synthetic aminoglycoside antibiotic. It is a new type of broad-spectrum antibiotic with a unique chemical structure and antibacterial mechanism of action. It not only has little cross-resistance with other antibiotics and antibacterial drugs, but also has a synergistic effect with most of them.
[0003] The structural formula of 3′,4′-dideoxy-kanamycin B is as follows: .
[0004] The master's thesis "Study on the Synthesis of Dibekacin Intermediates" (Wu Fangyuan, 2010, Zhengzhou University) disclosed the following synthesis route of dibekacin: Compound No. 2 in the above route, 1,3,2',6',3''-penta-N-p-toluenesulfonyl kanamycin B, is a key intermediate in the synthesis of 3',4'-dideoxy-kanamycin B. The structural formula of 1,3,2',6',3''-penta-N-p-toluenesulfonyl kanamycin B is as follows: .
[0005] The quality control of 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B has a positive impact on the quality and yield of the final product dibekacin. Summary of the invention
[0006] The technical problem to be solved by the present invention is to provide a rapid and accurate detection method suitable for 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B.
[0007] The technical solution to achieve the purpose of the present invention is a method for detecting an aminoglycoside drug intermediate, wherein the drug intermediate is 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B. The sample to be tested is first separated by an HPLC column and then detected by an ultraviolet-visible light detector.
[0008] The chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel packing column; The mobile phase is divided into an organic phase and an aqueous phase. The organic phase is acetonitrile and the aqueous phase is a 0.09% to 0.15% trifluoroacetic acid aqueous solution. The flow rate of the mobile phase is 0.8-1.1 mL / min; The injection volume is 10-20 μL; During elution, the volume ratio of the organic phase to the aqueous phase first gradually increases from 40%~20%:60%~80% to 60~80%:40%~20% within 10 minutes; after maintaining this ratio for 4 minutes, the ratio increases to 95%~75%:5%~25% within 10 minutes; after maintaining this ratio for 1 minute, the ratio slowly decreases to 40%~20%:60%~80% within 5 minutes; The detection wavelength of the UV-visible light detector is 220-240 nm.
[0009] Preferably, the particle size of the chromatographic column filler is 4-10 μm, and the column size is 150-250 mm×4.6-8.0 mm.
[0010] Preferably, the column temperature of the chromatographic column is 30-40°C.
[0011] As an option, gradient elution conditions are as follows: .
[0012] As an option, gradient elution conditions are as follows: .
[0013] As an option, gradient elution conditions are as follows: .
[0014] Optionally, the concentration of the aqueous trifluoroacetic acid solution in the aqueous phase is one of 0.09%, 0.1%, and 0.11%.
[0015] Optionally, the injection volume is one of 10 μL and 15 μL.
[0016] Preferably, the flow rate of the mobile phase is 1.0 mL / min.
[0017] Preferably, the detection wavelength of the UV-visible light detector is 228 nm.
[0018] The present invention has positive effects: (1) The present invention selects a specific mobile phase and a mobile phase injection method, the detection method has good specificity, is fast and accurate, and is suitable for the detection of 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B.
[0019] (2) Direct analysis of 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B as the sample can effectively separate the main component and impurities, with a limit of quantification of 0.3µg / mL; the linear range is 0.3µg / mL~900µg / mL; and the RSD of the main component repeatability is 0.23%.
[0020] (3) The detection limit of the detection method of the present invention is low, which ensures that the purity of 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B used in the synthesis is above a certain limit, thereby effectively ensuring the quality and yield of the final product dibekacin. BRIEF DESCRIPTION OF THE DRAWINGS
[0021] Figure 1 This is the HPLC spectrum of 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B detected in Example 1.
[0022] Figure 2 This is the HPLC spectrum of 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B detected in Example 2.
[0023] Figure 3 This is the HPLC spectrum of 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B detected in Example 3. DETAILED DESCRIPTION
[0024] The detection method of the dibekacin intermediate 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B of the present invention is as follows: firstly, the sample to be tested is separated by an HPLC column, and then detected by an ultraviolet-visible light detector.
[0025] The equipment, materials and conditions used are as follows: Chromatograph: Agilent 1260 series liquid chromatograph, equipped with UV-visible detector; ChemStation workstation.
[0026] Chromatographic column: octadecylsilane bonded silica gel filler chromatographic column, filler particle size 4-10μm, column specifications 150-250mm×4.6-8.0mm.
[0027] The mobile phase is divided into an organic phase and an aqueous phase. The organic phase is acetonitrile; the aqueous phase is a trifluoroacetic acid aqueous solution with a concentration of 0.09% to 0.11%.
[0028] The volume ratio of the organic phase to the aqueous phase is shown in the following table: .
[0029] The flow rate of the mobile phase is 0.8~1.1mL / min.
[0030] The column temperature is 30-40℃.
[0031] The injection volume is 10~20μL.
[0032] The detection wavelength of the UV-visible detector is 220~240nm.
[0033] (Example 1) The equipment, materials and conditions used in the detection method of the dibekacin intermediate 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B of this embodiment are as follows: Chromatograph: Agilent 1260 series liquid chromatograph, equipped with UV-visible detector; ChemStation workstation.
[0034] Chromatographic column: Octadecylsilane bonded silica gel packing column, column size 150mm×4.6mm, packing particle size 5μm.
[0035] The aqueous phase was 0.1% trifluoroacetic acid.
[0036] The volume ratio of organic phase to aqueous phase at different times is shown in the following table: .
[0037] The mobile phase flow rate was 1 mL / min.
[0038] The injection volume was 10 μL.
[0039] The detection wavelength of the UV-visible detector was 228 nm.
[0040] The dibekacin intermediate 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B to be tested was prepared into a 0.3 mg / mL solution and injected for detection. According to the above conditions, the HPLC spectrum obtained is shown in Figure 1 ,Depend on Figure 1 It can be seen that the detection method of this embodiment can effectively separate the main component 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B and related impurities; and adjacent substances can be completely separated.
[0041] Furthermore, the purity of the dibekacin intermediate 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B was calculated according to the area normalization method. The purity of 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B detected in this example was 95.9%.
[0042] (Example 2) The rest of the detection method of this embodiment is the same as that of the embodiment, except that: The column size is 250mm×4.6mm, and the filler particle size is 5μm.
[0043] The aqueous phase was 0.11% trifluoroacetic acid.
[0044] The volume ratio of organic phase to aqueous phase at different times is shown in the following table: .
[0045] The mobile phase flow rate was 1.0 mL / min.
[0046] The injection volume was 15 μL.
[0047] The dibekacin intermediate 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B to be tested was prepared into a 0.6 mg / mL solution and injected for testing. According to the above conditions, the HPLC spectrum obtained is shown in Figure 2 , test results: 1,3,2',6',3''-penta-N-p-toluenesulfonyl kanamycin B and related impurities all achieved baseline separation.
[0048] (Example 3) The rest of the detection method of this embodiment is the same as that of the embodiment, except that: The column size is 150mm×4.6mm, and the filler particle size is 5μm.
[0049] The aqueous phase was 0.09% trifluoroacetic acid.
[0050] The volume ratio of organic phase to aqueous phase at different times is shown in the following table: .
[0051] The mobile phase flow rate was 1.0 mL / min.
[0052] The injection volume was 15 μL.
[0053] The dibekacin intermediate 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B to be tested was prepared into a 0.6 mg / mL solution and injected for testing. According to the above conditions, the HPLC spectrum obtained is shown in Figure 3 , test results: 1,3,2',6',3''-penta-N-p-toluenesulfonyl kanamycin B and related impurities all achieved baseline separation.
[0054] (Example 4, Quantitative Limit of Detection Method) The dibekacin intermediate 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B was prepared into solutions of 0.075 µg / mL, 0.15 µg / mL and 0.3 µg / mL, and injected respectively according to the conditions of Example 1. The concentration of 0.3 µg / mL with a signal-to-noise ratio greater than 10:1 was selected as the quantitative limit, and the 0.3 µg / mL solution was injected 6 times continuously for analysis.
[0055] The signal-to-noise ratios of the six tests were all greater than 10:1, and the RSD was less than 10%.
[0056] (Example 5, linear range of detection method) Prepare standard curve solutions: 0.3µg / mL, 6µg / mL, 60µg / mL, 300µg / mL, 480µg / mL, 600µg / mL, 720µg / mL, 900µg / mL.
[0057] The peak area was recorded under the detection conditions of Example 1, and a peak area-concentration standard curve was drawn. The linear results are shown in the table: .
[0058] The linear equation is: y=22.318x+134.41, R 2 =0.9987.
[0059] (Example 6, Repeatability of Detection Method) The dibekacin intermediate 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B was prepared into a 0.6 mg / mL solution, and the solution was analyzed by continuous injection 6 times according to the detection conditions of Example 1.
[0060] The RSD of the principal component repeatability was 0.23%.
Claims
1. A method for detecting an aminoglycoside drug intermediate, wherein the drug intermediate is 1,3,2',6',3''-penta-N-toluenesulfonyl kanamycin B, characterized in that: The sample to be tested is first separated by HPLC column and then detected by UV-visible detector; The chromatographic conditions are as follows: Chromatographic column: octadecylsilane bonded silica gel packing column; The mobile phase is divided into an organic phase and an aqueous phase. The organic phase is acetonitrile and the aqueous phase is a 0.09% to 0.11% trifluoroacetic acid aqueous solution. The flow rate of the mobile phase is 0.8-1.1 mL / min; The injection volume is 10-20 μL; During elution, the volume ratio of the organic phase to the aqueous phase first gradually increases from 40%~20%:60%~80% to 60~80%:40%~20% within 10 minutes; after maintaining this ratio for 4 minutes, the ratio increases to 95%~75%:5%~25% within 10 minutes; after maintaining this ratio for 1 minute, the ratio slowly decreases to 40%~20%:60%~80% within 5 minutes; The detection wavelength of the UV-visible light detector is 220-240 nm.
2. The method for detecting an aminoglycoside drug intermediate according to claim 1, characterized in that: The particle size of the chromatographic column filler is 4 to 10 μm, and the column specifications are 150 to 250 mm × 4.6 to 8.0 mm.
3. The method for detecting an aminoglycoside drug intermediate according to claim 1, characterized in that: The column temperature is 30-40℃.
4. The method for detecting an aminoglycoside drug intermediate according to claim 1, characterized in that The gradient elution conditions were as follows: 。 5. The method for detecting an aminoglycoside drug intermediate according to claim 1, characterized in that The gradient elution conditions were as follows: 。 6. The method for detecting an aminoglycoside drug intermediate according to claim 1, characterized in that The gradient elution conditions were as follows: 。 7. The method for detecting an aminoglycoside drug intermediate according to claim 1, characterized in that: The concentration of the aqueous trifluoroacetic acid solution in the aqueous phase is one of 0.09%, 0.1% and 0.11%.
8. The method for detecting an aminoglycoside drug intermediate according to claim 1, characterized in that: The injection volume is either 10 μL or 15 μL.
9. The method for detecting an aminoglycoside drug intermediate according to claim 1, characterized in that: The mobile phase flow rate was 1.0 mL / min.
10. The method for detecting an aminoglycoside drug intermediate according to claim 1, characterized in that: The detection wavelength of the UV-visible detector was 228 nm.